Evaluation of Transgenes in Floral Crops for Arthropod Resistance 1996 Proposal
Evaluation of Transgenes in Floral Crops for Arthropod Resistance
John P. Sanderson, Assoc. Professor
Dept. of Entomology, Cornell University, Ithaca, NY 14853
Executive
Summary: Plants that can
defend themselves from arthropod infestations can
simplify pest control. Plant resistance is therefore a particularly attractive
least-toxic control
strategy for insect pest management on floral crops. However, the commercial
breeders that
produce a multitude of new varieties of floral crops each year must breed
for aesthetic and
horticultural qualities that directly sell plants, not for pest control.
It is also impractical to screen all
new cultivars for their relative resistance to arthropods each year. Futhermore,
because of the
diverse genetic systems of floral crop species it is nearly impossible
to introduce resistance into the
wide range of floral crops via traditional breeding methods. However, the
ability to insert novel
genes that code for products that are toxic to arthropod pests into floral
crops opens up a promising
way to create plant resistance. Once effective genes are discovered and
plant regeneration
techniques developed, it is conceivable that these genes may be inserted
into any number of crop
species and cultivars. Because floral crops are not eaten for food, there
is no need to determine
food tolerances for the gene products. Transgenic floral plants offer a
revolutionary method for
least-toxic pest control on ornamentals. My lab has been collaborating
with Sanford Scientific,
Inc. (SSI), based in nearby Waterloo, New York, to develop the first transgenic
floral crops with
the ability to resist arthropod infestations, and to evaluate their use
within an IPM context. SSI is
exclusively interested in using gene-gun technology to transform ornamental
crops.
The current project goal is to evaluate the use of transgenes in floral
crops for resistance to
greenhouse arthropod pests, using petunia and poinsettia as the initial
model systems. The initial
pest emphasis will be whiteflies, followed by other arthropod pests. Specifically,
we plan to: 1.
Screen gene products for biological activity against whitefly species affecting
greenhouse floral
crops (Bemisia argentifolii and Trialeurodes vaporariorum), using artificial
feeding assays, 2.
Transform petunia and poinsettia with genes that code for the biologically-active
products (done at
SSI); 3. Assess the transgenic plants for impact on whiteflies, and determine
correlation between
gene expression levels and levels of resistance; 4. Assess whitefly-resistant
transgenic petunia for
resistance to other greenhouse arthropod pests, including spider mites
(Terranychus urticae),
aphids
(e-g. Aphis gossypii, Myzus persicae), and thrips (Frankliniella occidentalis).
5. Assess
the effect
of transgenic plants on appropriate natural enemies (e.g. Encarsia formosa,
Phyroseiulus
persimilis).
Introduction
& Literature Review- Floral crops are sold for their beauty. Thus
there is a
zero-tolerance
for pests or their damage, which has led to multiple, routine pesticide
applications.
However,
pesticide resistance (Sanderson & Roush 1992), among other problems,
have made
growers
interested in cost-effective pest management tactics that maximize the
use of alternatives to
pesticides.
Plant resistance is a particularly attractive least-toxic control strategy
for floral crop IPM.
Plants
that can defend themselves from infestation can simplify pest control.
However, the
breeders
that produce hundreds of new varieties of floral crops each year select
for aesthetic and
horticultural
qualities that directly sell plants, not for pest control. It is impractical
to screen all new
cultivars
for arthropod resistance each year. Futhermore, because of the diverse
genetic systems of
floral
crop species it is nearly impossible to introduce resistance via traditional
breeding methods.
However,
the ability to insert novel genes that code for products that are toxic
to arthropod pests
into floral
crops opens up a promising way to create plant resistance. Once effective
genes are
discovered
and plant regeneration techniques developed, it is conceivable that these
genes may be
inserted
into any number of crop species and cultivars. Because floral crops are
not eaten for food,
there
is no need to determine food tolerances for the gene products. The Sanderson
lab is
collaborating
with Sanford Scientific, Inc. (SSI), based in upstate New York, to evalute
transgenes
for arthropod
resistance on floral crops. SSI is exclusively interested in using gene-gun
technology
to transform ornamental crops.
Furthermore, transgenic plants that provide insect resistance have almost
exclusively
utilized
toxins from Bacillus thuringiensis for chewing insect pests, and such toxins
are generally
not active
against phloem-feeding insects (Gasser & Fraley 1989, Gill et al. 1992,
Meeusen &
Warren
1989). Some work has been done with other genes, such as those that code
for protease
inhibitors including
trypsin inhibition (Hilder et al. 1987, Ryan 1989, Hoffmann et al. 1992),
and
chitinolytic
enzymes (against fungi - Broglie et al. 1991). But again, with the possible
exception
of chitinases,
these gene products generally are not very effective against phloem-feeders,
possibly
because
of differences in gut pH at which these enzymes are active (Berenbaurn
1990), differences
in internal
morphology and physiology among these insects, or because the guts of phloem-feeders
filter
nutrients from very dilute liquids, and they may not ingest an effective
amount of certain gene
products.
Because the most serious greenhouse insect pests are not leaf-chewing pests,
this
project
focuses specifically on novel gene products for control of sucking insect
pests such as
aphids
and whiteflies. Furthermore, because these gene products are likely to
be systemically
expressed
in the plant, other pests such as thrips and spider mites will also ingest
them and may
be controlled.
Genes that code for various other toxins (such as venoms) are available,
but these
gene products
are broken down in the insect gut and would have no effect via ingestion.
As a starting point, we are transforming petunias and poinsettias. Petunias
are an excellent
model
crop because they are attacked by a variety of greenhouse pests (including
whiteflies, thrips,
aphids,
and spider mites), transformation procedures are relatively simple, transgenic
plants are
recovered
rapidly, and they are a popalar bedding plant crop. Poinsettias are the
single most
valuable
potted floral crop and are severely attacked by Whiteflies (Byrne et al.,
1990).
Sanford Scientific, Inc. (SSI), based in upstate New York, is exclusively
licensed to use,
the gene-gun
process to transform ornamental crops (Dr. John Sanford is a co-inventor
of the gene-
gun).
SSI has developed effective transformation systems for petunia and poinsettia,
has licensed
a variety
of promising genes, and has already expressed some of these genes in petunia
and
poinsettia.
The Sanderson lab, in collaboration with SSI has done preliminary evaluations
of
some of
these gene products for effects on whiteflies, as follows.
Our initial (unpublished work has revealed a promising effect of tryptamine
on Bemisia
argentifolii
adult mortality in artifical feeding assays (Fig. 1). The indole alkaloid
tryptamine is an
analog
of the neurotransmitter scrotonin which is involved in animal nerve transmission.
Concentrations
of tryptamine in the plant are therefore likely to affect numerous insect
pests. A
gene isolated
from the plant Catharanthus roseus produces tryptamine decarboxylase (TdC),
which
converts tryptophan in the plant to tryptamine (Songstad et al. 1990).
Tryptamine also has
been reported
to cause reduced survival and deterrency against the aphid Rhopallosiphum
maidis
(Salem
1991).
GNA is a lectin gene from snowdrop plants (Galanthus nivalis), licensed
from Axis
Genetics,
Ltd. Extensive unpublished research has shown that the lectin produced
by the gene has
broad
anti-insect activity, especially against Homoptera. It has been reported
to reduce aphid
development,
perhaps by binding to the gut lining and eventually degrading the gut.
It generally
does not
cause acute toxicity, but affected aphids fail to thrive or reproduce.
We have not seen
acute
toxicity to adult silverleaf whitefly, but it is possible that nymphal
development and survival
may be
affected due to long-term ingestion during immature development.
SSI has transformed petunia and poinsettia with both the TdC and the GNA
gene, and
initial
whitefly screening studies of the transgenic plants are underway at Cornell.
The products produced by TdC and GNA genes have the potential to affect
numerous
arthropod
pests. Thus, transgenic plants that affect whiteflies are likely to affect
other insect pests
that feed
on phloem sap, such as aphids, mealybugs, and scale insects. Using an appropriate
promoter,
gene products can be expressed systemically throughout the plant, and thus
may affect
other
arthropods such as thrips and/or spider mites. Transgenic petunia lines
that affect whiteflies
will also
be screened against many of these other arthropods. Petunia rather than
poinsettia will be
used because
these other pests prefer petunia over poinsettia.
Furthermore, inserting both genes into the same plant should provide multiple
modes of
action,
and possibly synergism, that could minimize resistance problem.
We recognize that there is little reason to assume that insects are less
likely to develop
resistance
to bioengineered insecticides than to those applied by growers. It is likely
that complex
resistance
will not be achieved for several reasons, and may not even be desirable,
in order to
minimize
resistance selection pressure (Roush & Tabashnik 1990). Ultimately,
however,
resistance
management strategies will dictate that transgenic plants must be used
as a part of a total
IPM program.
Thus we also propose to evaluate the impact of transgenic plants on selected
natural
enemies
(Johnson & Gould 1992). Parasitoids and predators can be affected when
attacking prey
that have
ingested sublethal amounts of toxicants (Zhang & Sanderson 1990, Zchori-Fein
et al.
1994).
Future research may evaluate the way that transgenic plants may be planted
in a greenhouse
to manage
possible resistance problems (e. a., Gould et al. 1994).
Objectives
& Anticipated Benefits: The project objectives are as follows:
1. Screen gene
products
for biological activity against whitefly species affecting greenhouse floral
crops (Bemisia
argentifolii
and Trialeurodes vaporariorum), using artificial feeding studies (some
of this work
has already
been accomplished). 2. Transform petunia and poinsettia with genes that
code for the
biologically-active
products (done at SSI). 3. Assess the transgenic plants for impact on whiteflies,
and determine
correlation between gene expression levels and levels of resitstance. 4.
Assess
transgenic
petunia with whitefly resistance for resistance to other greenhouse arthropod
pests,
including
spider mites (Tetranychus urticae), aphids (e.g. Aphis gossypii, Myzus
persicae), and
thrips
(Frankliniella occidentalis). 5. Assess the effect of transgenic plants
on appropriate natural
enemies
(e.g. Encarsia formosa, Phytoseiulus persimilis).
Transgenic floral plants offer a revolutionary method for pesticide-free
pest management on
floral
crops. Success in this research could also identify some of the first gene
products that are
effective
against plant-sucking pests. These proposed studies use petunia and poinsettia
as a model
system; effective
genes may then be inserted into numerous other floral crops, bypassing
the
constraints of traditional
plant-breeding efforts for insect resistance in floral crops.
Materials &
Methods:
Objective 1,
SSI is currently evaluating genes and gene products from a variety of sources,
including a Trichoderma
chitinase from Dr. Gary Harmon’s Cornell lab (which should attack the
chitin-based lining
of the arthropod gut), a series of lyric peptides designed by Demeter
Bio/Technologies (which
should disrupt the membranes of epithelial cells lining the arthropod gut),
and a proprietary
gene from commercial sources, which cannot be disclosed in this format
at this
time. In each case,
the relevant gene product (ie. synthetic peptide, purified chitinase, etc.)
will be
prepared and incorporated
into an artificial feeding assay for adult whiteflies (e.g., Fig. 1). The
test
products will be serially
diluted in a 10% sucrose solution, and a small amount (0.5 ml) will be
placed between two
thin pieces of parafilm stretched over the open top of plastic vials, serving
as a
feeding membrane.
Twenty newly-eclosed adult whiteflies will be placed into each vial. Assays
will be done at 25C,
and 14 h light:10 h dark. Each assay will have three replicates of each
treatment (including
a 10% sucrose control), and all experiments repeated three times. Insect
morality will be recorded
after 12, 24, 48, and 72 hours. The feasibility of achieving in plants
concentrations at
which each product had activity will be assessed before attempting the
next step.
Gene products will
be assayed individually and in combination to evaluate possible synergy.
Objective 2.
Genes with the most promising gene products will be cloned behind strong
constituitive plant
promoters, and will be biolistically delivered into petunia and poinsettia
using
procedures already
developed at SSI. Selection procedures will be based upon Hygromycin
resistance, already
developed at SSI. Two promising genes, GNA and TdC, have already been
prepared and delivered
into petunia, such that some transgenic plants are currently available
for
testing. SSI will
produce numerous plants of each type of transformation, providing large
enough
samples to assess
each gene inspite of variability in transgenic expression levels. This
work will
be done by SSI and
no funds are requested for this work.
Objective 3.
Preliminary screening will be done at SSI to identify the degree of gene
product
expression for each
line of plants for each gene. Promising plants will then undergo preliminary
screening for
whitefly resistance (both greenhouse whitefly and silverleaf whitefly)
at Cornell.
Promising
plants will then undergo more exacting analysis for degree and nature of
resistance, and
will be
further analyzed for exact gene expression level to determine correlation
between gene
expression
level and level of resistance. No-choice tests will be done in a growth
chamber (25C,
L:D 14:10,
50% RED to assess the degree of whitefly resistance expressed in the transformed
plants.
For petunia, test leaves will be rinsed with methanol or ethanol to remove
the acylsugars
contained
in the glandular trichomes. Aclysugars can be toxic to arthropods (Liedl
et al. 1995).
Two-to-five
day-old adult whiteflies of a single species will be caged on individual
leaves of
similar
age from the middle of the canopy within clip-rages. Adult survival will
be scored after six
days,
and the resulting number of eggs counted. Thereafter, cohorts of individual
nymphs will be
observed
daily on each leaf to assess immature mortality and developmental time
on transformed
versus
non-transformed plants. At least three single-leaf replicates of each transgenic
line, plus a
non-transformed
control line, will be used in each experiment. Each experiment will be
done a
minimum
of three times. Assays for the level of gene product(s) expression in the
leaves will be
done it
the beginning and end of each experiment. In other tests, whitefly assays
as described
above
will be done on leaves of various ages from plants of the most promising
transgenic lines,
plus a
control line. Concentrations of the gene product(s) in leaves of various
ages and canopy
heights
will be assessed and correlated with insect survival and development to
determine the
degree
of variation of within-plant resistance.
Objective
4. Transgenic petunia lines with significant whitefly resistance will
be used to assess
resistance
to other greenhouse arthropod pests, including spider mites (Tetranychus
urticae),
aphids
(e.g. Aphis gossypii, Myzus persicae), and thrips; (Frankliniella occidentalis).
No-choice
tests,
similar to those described in Objective 3 for whiteflies, will be performed
for these additional
major
jests, using appropriate techniques to confine arthropods on leaves (e.g.,
clip cages,
dialysis
tubing cages, Munger sells, etc.) (Oi et al. 1989, Mang & Sanderson
1990).
Objective
5. The effect of transgenic plants on a common greenhouse arthropod
predator and a
parasitoid
(e.g. Phytoseiulus persimilis, Encarsia formosa) will be assessed by confining
adult
natural
enemies on transformed and non-transformed plants and measuring their survival.
To
evaluate
any possible toxic effect of the transgenic leaf alone, adult natural enemies
of standardized
age will
be confined on methanol-washed uninfested leaves of the transgenic lines
with the highest
resistance,
plus a control, and checked for survival after 24 h. For P. persimilis,
methodology
using
leaf disks on wet cotton in petri dishes from Zhang & Sanderson (1990)
will be modified for
use with
transgenic plants rather than an acaricide. For E. formosa, wasps will
be pre-fed with
honey
before being confined on the leaves in clip-cages. To evaluate whether
the natural
enemies
are affected by feeding on prey that are likely to be intoxicated, transgenic
lines that
express
a high level of gene product but also allow some pest survival will be
used. For P.
persimilis,
methodology similar to Zhang & Sanderson (1990) will be used. For E.
formosa,
individual
wasps will be confined on washed leaves in the presence of >25 third instar
whitefly
nymphs
for 24 h and checked for survival. Thereafter the wasps will be eliminated
and whitefly
nymphs
will be held and checked for amount of successful parasitism (wasp exit
holes) and
evidence
of host-feeding, relative to non-transformed controls (Zchori-Fein et al.
1994). Each
test will
be replicated at least three times. Test leaves will be assayed for the
level of gene product
at the
end of each test.
Literature
Cited:
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M. 1980. Adaptive significance of midgut pH in larval Lepidoptera. Amer.
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115: 138-146.
Broglie,
K., I. Chet, M. Holliday, R. Cressman, P. Biddle, S. Knowlton, C.J. Mauvais,
& R.
Brodlie. 1991. Transgenic plants with enhanced resistance to the fungal
pathogen
Rhizoctonia solani. Science 254: 1194-1197.
Byrne,
D.N., T.S. Bellows, & M.P. Parrella. 1990. pp. 227-261. In: D. Gerling
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Gasser,
C.S. & R.T. Fraley. 1989. Genetically engineering plants for crop improvement.
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Gill,
S.S., E.A. Cowles, & P.V. Pietrantonio. 1992. The mode of action of
Bacillus
thuringiensis endotoxins. Ann. Rev. Entomol. 37: 615-636.
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F., P. Follett, B. Nault, & G. Kennedy. 1994. Resistance management
strategies for
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R.K. Jannson, M.L. Powelson, & K.V. Raman, eds. APS Press, St. Paul,
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(Lepidoptera: Noctuidae). 1. Econ. Entomol. 85: 2516-2522.
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M.T. & F. Gould. 1992. Interactions of genetically engineered host-plant
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tobacco. Environ.
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Budget:
Personnel:
Graduate Research Associate
$19,964
Expenses:
Walk-in growth chamber charges* (12 months @ S60/mo.)
$720
Greenhouse space charges* ( 600 sqft @ $2.40/sq.ft./yr)
$1,440
Miscellaneous supplies (pots, fertilizer, growing media, chemicals, cages)
$700
Total:
$22,824
Due to college/departmental budget cutbacks, faculty projects must provide
funds for use of
greenhouse and growth chamber space.
Note: The New York State Center for Advanced Technology - Biotechnology
Program has
provided initial funds for technical support and some supplies for this
project through 6/96.
Funding for technical support and some supplies will again be sought from
the Biotechnology
Program. The Kenneth Post Foundation provided funds ($1,863) for supplies
to bring available
greenhouse facilities into compliance with Standard Biosafety Level P-1
for research with
transgenic plants (NIH Recombinant DNA Guidelines, 5/7/86, Appendix G-11-a,
p. 16972-
16973;
Federal Register, 8/11/87, p. 29804).
Leader
Qualifications:
Dr. John Sanderson is Associate Professor of Entomology at Cornell
University with
research (50%) and extension
(50%) responsibilities on insect and mite pest management on
greenhouse floral crops.
He has been involved in insect pest management on greenhouse floral
crops since joining the
Entomology faculty at Cornell University in 1987. He received his B.S.
degree in Zoology at San
Diego State University, and M.S. and Ph.D. degrees in Entomology at
the University of California,
Riverside. He then completed a postdoctoral position with Dr.
Michael Parrella at UC Riverside
prior to coming to Cornell. He was promoted to Associate
Professor with tenure in
1994. He has published numerous scientific and extension articles, and
has been an invited speaker
at numerous industry conferences, including most of the SAF
Conferences on Insect and
Disease Management on Ornamentals. He has organized a Greenhouse
IPM Implementation Workgroup
in the Northeastern U.S. for regional researchers and extension
professionals from 10 states/provinces.
He is an integral member of Cornell’s Greenhouse IPM
Team, and of Cornell’s effort
in Controlled Environment Agriculture.
